Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P11/00—Preparation of sulfur-containing organic compounds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/12—Methionine; Cysteine; Cystine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/15—Corynebacterium

Definitions

• the invention relates to a new process for the fermentative production of sulfur-containing fine chemicals, in particular L-methionine and L-cysteine, in which bacteria are used in which nucleotide sequences are expressed which are mutants of S-adenosylmethionine synthase (metK) (EC2.5. 1.6) encode; Nukeotide sequences which code for these mutants, as well as the recombinant microorganisms transformed with them, and novel metK mutants with changed enzyme activity.
• metalK S-adenosylmethionine synthase
• Sulfur-containing fine chemicals such as, for example, methionine, homocysteine, S-adenosyl-methionine, glutathione, cysteine, biotin, thiamine, lipoic acid
• These substances collectively referred to as "sulfur-containing fine chemicals"
• JP-A-06-020809 discloses a nucleotide sequence for a gene coding for S-adenosylmethionine from Brevibacterium flavum MJ-233, a coryneform bacterium.
• the corresponding amino acid sequence comprises 412 amino acids.
• positions 24 and 94 among other things, the protein each has a cysteine residue which is conserved in the corresponding enzymes of numerous other coryneform bacteria.
• the amino acid sequence disclosed has a characteristic sequence section between residues 137 and 154. The production of mutants and their use in the fermentative production of sulfur-containing fine chemicals is not described therein
• a metK gene from C. glutamicum is known from WO-A-01/00843, which codes for a protein with 407 amino acids and has a sequence according to SEQ ID NO: 16.
• Improvements in the fermentative production of fine chemicals usually correlate with improvements in material flows and yields. It is important to prevent or reduce intermediate or end product inhibitions of important synthetic enzymes. It is also advantageous to prevent or reduce outflows of the carbon flow into undesired products or side products.
• S-adenosylmethionine An important metabolite of methionine and thus an essential outflow is S-adenosylmethionine.
• S-adenosylmethionine is also a crucial regulator of methionine biosynthesis.
• S-adenosylmethionine acts there as a co-repressor of the repressor metJ (Weissbach, H. Brot, N. (1991) Mol Microbiol. 5 (7), 1593-1597).
• S-adenosylmethionine is also an essential outflow of the desired product of value L-methionine. Therefore, it is desirable to reduce the amount of S-adenosylmethionine formed for a number of reasons: a) the amount of L-methionine formed would be increased, b) the repression of genes of methionine biosynthesis would be reduced, and c) the feedback inhibition of enzymes methionine biosynthesis would be reduced.
• metK is described as an essential gene and thus appears to the person skilled in the art as a starting point for improved fermentative production of sulfur-containing fine chemicals, in particular L-methionine.
• the object of the invention is therefore to provide a new process for the improved fermentative production of sulfur-containing fine chemicals, in particular L-methionine, and the means required for this.
• the above object is surprisingly achieved by providing a process for the fermentative production of a sulfur-containing fine chemical, comprising the expression of a metK nucleotide sequence in a coryneform bacterium, the nucleotide sequence coding for an S-adenosylmethionine synthase mutant, the activity of which changes, preferably reduced, compared to the wild-type enzyme, is.
• the mutant is derived from the S-adenosylmethionine synthase from Corynebacterium glutamicum and, when measured in Corynebacterium glutamicum, shows less activity than the wild-type enzyme.
• a first subject of the invention relates to a process for the fermentative production of at least one sulfur-containing fine chemical, which comprises the following steps: a) fermentation of a coryneform bacterial culture producing the desired sulfur-containing fine chemical, wherein in the coryneform bacteria at least one nucleotide sequence is expressed which codes for a protein with altered S-adenosylmethionine synthase (metK) activity; b) accumulation of the sulfur-containing fine chemical in the medium and / or in the cells of the bacteria, and c) isolation of the sulfur-containing fine chemical, which preferably comprises L-methionine.
• the mutated coryneform bacterium also has, compared to the non-mutated wild type, improved metY activity and / or an increased amount of L-methionine (e.g. in g / l fermentation broth).
• a coding nucleotide sequence which codes for a protein with reduced metK activity, in which at least one cysteine residue of the wild-type protein is substituted, is used in particular as the coding nucleotide sequence.
• the metK-coding sequence is preferably a coding nucleotide sequence which codes for a protein with metK activity which has the following partial amino acid sequence as shown in SEQ ID NO: 23:
• the metK coding sequence used according to the invention preferably comprises a coding sequence according to SEQ ID NO: 21 or a nucleotide sequence homologous thereto which codes for a protein with metK activity.
• coryneform bacteria are fermented, in which at least one of the genes selected from genes of the above-mentioned group 1) to 18) is mutated at the same time in such a way that the corresponding proteins are less or less than non-mutated proteins are not influenced in their activity by metabolic metabolites and that in particular the production of the fine chemical according to the invention is not impaired or so that their specific enzymatic activity is increased:
• coryneform bacteria are fermented in which at least one of the genes selected at the same time is selected
• the gene lysA coding for the diaminopicolinate decarboxylase is weakened, in particular by reducing the expression rate of the corresponding gene.
• coryneform bacteria are fermented in which at least one of the genes from groups 19) to 28) above is mutated at the same time, so that the enzymatic activity of the corresponding protein is partially or completely reduced.
• Microorganisms of the type Corynebacterium glutamicum are preferably used in the process according to the invention.
• the invention further relates to a method for producing an L-5 methionine-containing animal feed additive from fermentation broths, which comprises the following steps: a) Cultivation and fermentation of an L-methionine-producing microorganism, preferably with reduced metK activity as defined above, in a fermentation medium; B) removal of water from the fermentation broth containing L-methionine; c) removal of the biomass formed during the fermentation in an amount of 0 to 100% by weight; and d) drying the fermentation broth obtained according to b) and / or c) in order to obtain the animal feed additive in the desired powder or granule form.
• the invention also relates to recombinant coryneform bacteria which express a mutated metK gene as defined above and in particular to those recombinant coryneform bacteria which no longer express the metK wild-type enzyme.
• Preferred recombinant coryneform bacteria show at least one of the following features in comparison to the corresponding wild-type strain: a) lower intracellular S-adenosylmethionine titer (b) lower intracellular S-adenosylmethionine synthase concentration, or c) lower activity of the S-adenosylmethionine synthase, determined on the basis of the S -Adenosylmethionine formation rate; and additionally optionally at least one of the following features: d) improved metY activity, or e) increased amount of L-methionine.
• Proteins with the biological activity of "S-adenosylmethionine synthase", also called metK for short (EC2.5.1.6), are those proteins which are capable of converting L-methionine and ATP to S-adenosyl-methionine Further details of the metK protein are known, and the enzymatic activity of metK can be demonstrated by enzyme tests, for instructions on this, see: Markham, GD et al. (1983) Methods in Enzymology 94: 219-222.
• sulfur-containing fine chemical encompasses any chemical compound which contains at least one sulfur atom covalently bound and is accessible by a fermentation process according to the invention.
• Non-limiting examples thereof are methionine, homocysteine, S-adenosyl-methionine, cysteine, and in particular methionine and S-adenosyl methionine.
• L-methionine also include the corresponding salts, such as, for. B. methionine hydrochloride or methionine sulfate.
• polypeptides are understood to mean peptides or proteins which contain two or more amino acids linked via peptide bonds.
• metabolic metabolite denotes chemical compounds which occur in the metabolism of organisms as intermediates or end products and which, in addition to their property as chemical building blocks, can also have a modulating effect on enzymes and their catalytic activity. It is known from the literature that such Metabolic metabolites can have both inhibitory and stimulating effects on the activity of enzymes (Biochemistry, Stryer, Lubert, 1995 WH Freeman & Company, New York, New York.) The literature also describes that this can be done by measures such as mutation genomic DNA by UV radiation, ionizing radiation or mutagenic substances and subsequent selection for certain
• weaken and “decrease” in the context of the invention describe the weakening or reduction of the intracellular activity of one or more enzymes in a microorganism which are encoded by the corresponding DNA. This can be done, for example, by deleting a gene in an organism, replacing an existing gene with another gene, reducing the copy number of a transcript of the gene or genes, using a weak promoter or using a gene which is suitable for a corresponding enzyme with a lower activity encoded and you can combine these measures if necessary.
• a “reduced activity” according to the invention is particularly given when the specific activity of the mutant is reduced to a residual activity of about 1 to 90%, preferably 3 to 70%, such as 5 to 10% of the wild-type activity.
• polynucleotide sequences according to the invention code for proteins with modified, in particular reduced, S-adenosylmethionine synthase activity as defined above.
• Cys24 and or Cys94 are different from one of Cys
• “Functional equivalents” naturally also include polypeptides that are accessible from other organisms, as well as naturally occurring variants. For example, regions of homologous sequence regions can be determined by sequence comparison and equivalent enzymes can be determined based on the specific requirements of the invention.
• Fusion proteins are also fusion proteins which have one of the abovementioned polypeptide sequences or functional equivalents derived therefrom and at least one further, functionally different, heterologous sequence in functional N- or C-terminal linkage (ie without mutual substantial functional impairment of the fusion protein parts)
• heterologous sequences are, for example, signal peptides, enzymes, immunoglobulins, surface antigens, receptors or receptor ligands.
• M / 43138 "Functional equivalents" encompassed according to the invention are homologs to the specifically disclosed proteins. These have at least 20%, 30%, or about 40%, 50%, preferably at least about 60%, 65%, 70%, or 75%, in particular at least 85 %, such as 90%, 95% or 99%, homology to one of the specifically disclosed sequences, calculated according to the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sei. (USA) 85 (8), 1988, 2444- 2448th
• X 3 represents a mutation-introduced amino acid, in particular alanine, which is different from Cys.
• X 3 corresponds to Cys94 of the metK 5 wild-type sequence of C. glutamicum (SEQ ID NO: 16).
• X 2 preferably represents Ala, Glu, Asp, Asn or Arg; and
• X 1 preferably represents Gly, Cys, Ser or Ala.
• homologs of the proteins or polypeptides of the invention can be generated by mutagenesis, e.g. by point mutation or shortening of the protein.
• the term: "homolog” as used here refers to a variant form of the protein that acts as an agonist or antagonist of protein activity.
• the invention relates both to isolated nucleic acid molecules which code for polypeptides or proteins or biologically active sections thereof, and to nucleic acid fragments which e.g. can be used as hybridization probes or primers for the identification or amplification of coding nucleic acids according to the invention.
• nucleic acid molecules according to the invention can also contain untranslated sequences from the 3 'and / or 5' end of the coding gene region
• nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid and, moreover, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or free of chemical precursors or other chemicals be when it's chemically synthesized.
• the invention further comprises the nucleic acid molecules complementary to the specifically described nucleotide sequences or a section thereof.
• the nucleotide sequences according to the invention enable the generation of probes and primers which can be used for the identification and / or cloning of homologous sequences in other cell types and organisms.
• probes or primers usually comprise a nucleotide sequence region which, under stringent conditions, can be attached to at least about 12, preferably at least about 25, e.g. about 40, 50 or 75 successive nucleotides of a sense strand of a nucleic acid sequence according to the invention or a corresponding antisense strand are hybridized.
• nucleic acid sequences which comprise so-called silent mutations or which have been modified in accordance with the codon usage of a specific source or host organism, in comparison to a specifically named sequence, as well as naturally occurring variants, such as e.g. Allelic variants, of which. Sequences obtainable also by conservative nucleotide substitutions (i.e. the amino acid in question is replaced by an amino acid of the same charge, size, polarity and / or solubility).
• the invention also relates to the molecules derived from the specifically disclosed nucleic acids by sequence polymorphisms. These genetic polymorphisms can exist between individuals within a population due to natural variation. These natural variations usually cause a 1 to 5% variance in the nucleotide sequence of a gene.
• the property of being able to “hybridize” to polynucleotides is understood to mean the ability of a poly- or oligonucleotide to bind to an almost complementary sequence under stringent conditions, while under these conditions non-specific bonds between non-complementary partners are avoided.
• the sequences should be closed 70-100%, preferably 90-100%, of complementary nature of complementary sequences to be able to specifically bind to each other
• metK genes coding for the enzyme S-adenosylmethionine synthase (EC 2.5.1.6) can be isolated in a manner known per se.
• cosmids such as the cosmid vector SuperCos I (Wahl et al. (1987), Proceedings of the National Academy of Sciences USA 84: 2160-2164), but also plasmids, such as pBR322 (Bolivar; Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268).
• Particularly suitable hosts are E. coli strains which are defective in terms of restriction and recombination.
• An example of this is the DH ⁇ mcr strain, which was described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649).
• the long DNA fragments cloned with the aid of cosmids can then in turn be subcloned into common vectors suitable for sequencing and subsequently sequenced
• coryneform bacteria whose reduced metK activity can be demonstrated by at least one of the following properties: a) a lower intracellular S-adenosylmethionine titer compared to the wild-type strain; b) a lower intracellular S-adenosylmethionine synthase concentration (less S-adenosylmethionine synthase based on total protein); or c) a lower intracellular S-adenosylmethionine synthase activity (less S-
• microorganisms serving as host cells in particular coryneform bacteria, which contain a vector, in particular a pendulum vector or plasmid vector, which carries at least one metK gene according to the invention, or in which a metK gene according to the invention is expressed with reduced activity.
• a vector in particular a pendulum vector or plasmid vector, which carries at least one metK gene according to the invention, or in which a metK gene according to the invention is expressed with reduced activity.
• microorganisms can produce sulfur-containing fine chemicals, in particular L-methionine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol.
• These are preferably coryneform bacteria, in particular of the genus Corynebacterium. From the genus Corynebacterium, the species Corynebacterium glutamicum should be mentioned in particular, which is known in the art for its ability to produce L-amino acids.
• KFCC Korean Federation of Culture Collection
• ATCC American Type Culture Collection
• Enzymes can be influenced in their activity by mutations in the corresponding genes in such a way that there is a partial or complete reduction in the reaction rate of the enzymatic reaction. Examples of such mutations are known to the person skilled in the art (Motoyama H. Yano H. Terasaki Y. Anazawa H. Applied & Environmental Microbiology. 67: 3064-70, 2001, Eikmanns BJ. Eggeling L. Sahm H.
• sulfur-containing fine chemicals in particular L-methionine
• coryneform bacteria in addition to expression of a metK gene according to the invention, one or more enzymes of the respective biosynthetic pathway, the cysteine metabolic pathway, and aspartate semialdehyde synthesis , glycolysis, anaplerotic, pentose-phosphate metabolism, the citric acid cycle or the export of amino acids.
• sulfur-containing fine chemicals in particular L-5 methionine
• M / 43138 to be built in.
• Inducible promoters also make it possible to increase expression in the course of fermentative L-methionine production. Expression is also improved by measures to extend the life of the mRNA. Furthermore, preventing the breakdown of the enzyme protein also increases the enzyme activity.
• the genes or gene constructs can either be present in plasmids with different copy numbers or can be integrated and amplified in the chromosome. Alternatively, overexpression of the genes in question can also be achieved by changing the media composition and culture management.
• the invention therefore also relates to expression constructs containing, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for a polypeptide according to the invention; and vectors comprising at least one of these expression constructs.
• Such constructs according to the invention preferably comprise a promoter 5'-upstream of the respective coding sequence and 3'-downstream a terminator sequence and, if appropriate, further customary regulatory elements, in each case operatively linked to the coding sequence.
• An “operative linkage” is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can perform its function as intended in the expression of the coding sequence.
• sequences which can be linked operatively are activation sequences and Enhancers, etc.
• Other regulatory elements include selectable markers, amplification signals, origins of replication, etc. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
• the natural regulatory sequence can still be present before the actual structural gene. This natural regulation can possibly be switched off by genetic modification and the expression of the genes increased or decreased.
• the gene construct can also have a simpler structure, ie no additional regulation signals are inserted in front of the structural gene and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated so that regulation no longer takes place and gene expression is increased or decreased.
• the nucleic acid sequences can be contained in one or more copies in the gene construct.
• promoters examples include: the promoters, ddh, amy, lysC, dapA, lysA from Corynebacterium glutamicum, but also gram-positive promoters, such as SPO2 as described in Bacillus Subtilis and Its Closest Relatives, Sonenshein, Abraham L., Hoch, James A., Losick, Richard; ASM Press, District of Columbia, Washington and Patek M. Eikmanns BJ. Patek J. Sahm H. Microbiology.
• inducible promoters such as, for example, light-inducible and in particular temperature-inducible promoters, such as the P r P r promoter.
• inducible promoters such as, for example, light-inducible and in particular temperature-inducible promoters, such as the P r P r promoter.
• all natural promoters with their regulatory sequences can be used.
• synthetic promoters can also be used advantageously.
• the regulatory sequences mentioned are intended to enable the targeted expression of the nucleic acid sequences. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
• the regulatory sequences or factors can preferably negatively influence the expression and thereby reduce it. Attenuation at the transcription level can take place by using weak transcription signals such as promoters and / or "enhancers". In addition, a weakening of the translation is also possible, for example, by reducing the stability of the mRNA.
• the regulatory sequences or factors can preferably have a positive influence on the expression and thereby increase or decrease it.
• the regulatory elements can advantageously be strengthened at the transcription level by
• M / 43138 strong transcription signals such as promoters and / or enhancers are used.
• an increase in translation is also possible, for example, by improving the stability of the mRNA.
• An expression cassette is produced by fusing a suitable promoter, a suitable Shine-Dalgamo sequence with a metK nucleotide sequence and a suitable termination signal.
• a suitable promoter for this purpose, common recombination and cloning techniques are used, such as, for example, in Current Protocols in Molecular Biology, 1993, John Wiley & Sons, Incorporated, New York New York, PCR Methods, Gelfand, David H., Innis, Michael A., Sinsky, John J. 1999, Academic Press, Incorporated, California, San Diego,., PCR Cloning Protocols, Methods in Molecular Biology Ser., Vol. 192, 2nd ed., Humana Press, New Jersey, Totowa. T. Maniatis, E.F.
• the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which enables optimal expression of the genes in the host.
• Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P.H. et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).
• vectors are also understood to mean all other vectors known to the person skilled in the art, such as phages, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be replicated autonomously in the host organism or can be replicated chromosomally.
• MetK genes according to the invention are expressed by way of example using episomal plasmids.
• Suitable plasmids are those which are replicated in coryneform bacteria.
• Numerous known plasmid vectors such as. B. pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl (Eikmanns et al., Gene 102: 93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107: 69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1.
• Other plasmid vectors such as. B.
• pCLiK5MCS SEQ ID NO: 9, or those based on pCG4 (US-A 4,489,160) or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)) or pAG1 (US-A 5,158,891) can be used in the same way.
• Such plasmid vectors are also suitable, by means of which the method of gene amplification by integration into the chromosome can be used, as described, for example, by Remscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for the duplication or amplification of the hom-thrB operon.
• the complete gene is cloned into a plasmid vector that can replicate in a host (typically E. coli) but not in C. glutamicum.
• vectors examples include pSUP301 (Simon et al., Bio / Technology 1,784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145,69-73 (1994)), Bernard et al., Journal of Molecular Biology , 234: 534-541 (1993)), pEM1 (Schrumpf et al. 1991, Journal of Bacteriology 173: 4510-4516) or pBGS8 (Spratt et al., 1986, Gene 41: 337-342) in question.
• Other plasmid vectors such as. B. pCLiK5MCS integrative sacB, SEQ ID NO: 12 can be used in the same way.
• the plasmid vector containing the gene to be amplified is then transformed into the desired strain of C. glutamicum by transformation.
• Methods for transformation are described, for example, by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Biotechnology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)).
• the microorganisms produced according to the invention can be cultured continuously or batchwise in the batch process (batch cultivation) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the production of sulfur-containing fine chemicals, in particular L-methionine.
• batch cultivation batch cultivation
• feed process feed process
• repetitive feed process repetition feed process
• a summary of known cultivation methods can be found in the textbook by Chmiel (Bioprocess Technology 1st Introduction to Biochemical Engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (bioreactors and peripheral devices (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)) Find.
• the culture medium to be used has to meet the requirements of the respective strains in a suitable manner. Descriptions of culture media of various microorganisms are contained in the manual "Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington DC, USA, 1981).
• These media which can be used according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.
• M / 43138 Preferred carbon sources are sugars, such as mono-, di- or polysaccharides.
• sugars such as mono-, di- or polysaccharides.
• very good carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
• Sugar can also be added to the media through complex compounds such as molasses or other by-products of sugar refining. It can also be advantageous to add mixtures of different carbon sources.
• Other possible carbon sources are oils and fats such as. B. Soybean oil. Sunflower oil.
• Peanut oil and coconut fat fatty acids such as As palmitic acid, stearic acid or linoleic acid, alcohols such as. B. glycerol, methanol or ethanol and organic acids such as. B. acetic acid or lactic acid.
• fatty acids such as As palmitic acid, stearic acid or linoleic acid
• alcohols such as. B. glycerol, methanol or ethanol
• organic acids such as. B. acetic acid or lactic acid.
• Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds.
• Exemplary nitrogen sources include ammonia gas or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract and others.
• the nitrogen sources can be used individually or as a mixture.
• Inorganic salt compounds that may be included in the media include the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
• Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, but also organic sulfur compounds, such as mercaptans and thiols, can be used as the sulfur source for the production of sulfur-containing fine chemicals, in particular methionine.
• Phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus.
• Chelating agents can be added to the medium to keep the metal ions in solution.
• Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
• the fermentation media used according to the invention usually also contain other growth factors, such as vitamins or growth promoters, for example
• M / 43138 Biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine include.
• Growth factors and salts often come from complex media components such as yeast extract, molasses, corn steep liquor and the like. Suitable precursors can also be added to the culture medium. The exact composition of the media connections strongly depends on the respective experiment and is decided individually for each specific case. Information about media optimization is available from the textbook "Applied Microbiol. Physiology, A Practical Approach” (Ed. PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like.
• All media components are sterilized either by heat (20 min at 1.5 bar and 121 ° C) or by sterile filtration.
• the components can be sterilized either together or, if necessary, separately. All media components can be present at the beginning of the cultivation or can be added continuously or in batches.
• the temperature of the culture is normally between 15 ° C and 45 ° C, preferably 25 ° C to 40 ° C and can be kept constant or changed during the experiment.
• the pH of the medium should be in the range from 5 to 8.5, preferably around 7.0.
• the pH for cultivation can be checked during the cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid.
• anti-foaming agents such.
• B. fatty acid polyglycol esters can be used.
• suitable selectively acting substances such as, for. B. antibiotics.
• oxygen or oxygen-containing gas mixtures such as. B.
• the temperature of the culture is usually 20 ° C to 45 ° C and preferably 25 ° C to 40 ° C.
• the culture is continued until a maximum of the desired product has formed. This goal is usually achieved within 10 hours to 160 hours.
• the fermentation broths obtained in this way in particular containing L-methionine, usually have a dry matter of 7.5 to 25% by weight.
• M / 43138 It is also advantageous if the fermentation is carried out with limited sugar at least at the end, but in particular for at least 30% of the fermentation period. This means that the concentration of usable sugar in the fermentation medium is kept to> 0 to 3 g / l or reduced during this time.
• the fermentation broth is then processed further.
• the biomass can be wholly or partially separated by methods such as. B. centrifugation, filtration, decanting or a combination of these methods can be removed from the fermentation broth or left completely in it.
• the fermentation broth using known methods, such as. B. with the help of a rotary evaporator, thin film evaporator, falling film evaporator, by reverse osmosis, or by nanofiltration, thickened or concentrated.
• This concentrated fermentation broth can then be worked up by freeze drying, spray drying, spray granulation or by other processes.
• the product-containing broth is subjected to chromatography with a suitable resin after the biomass has been separated off, the desired product or the impurities being wholly or partly retained on the chromatography resin.
• chromatography steps can be repeated if necessary using the same or different chromatography resins.
• the person skilled in the art is skilled in the selection of the suitable chromatography resins and their most effective application.
• the purified product can be concentrated by filtration or ultrafiltration and kept at a temperature at which the stability of the product is maximum.
• the identity and purity of the isolated compound (s) can be determined by prior art techniques. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin-layer chromatography, NIRS, enzyme tests or microbiological tests. These analysis methods are summarized in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp.
• Figure 1 shows the results of a radioactive metK assay, carried out with wild-type enzyme or C94A mutant.
• ampicillin resistance and origin of replication of the vector pBR322 were amplified with the oligonucleotide primers SEQ ID NO: 1 and SEQ ID NO: 2 using the polymerase chain reaction (PCR).
• the oligonucleotide primer SEQ ID NO: 1 in 5'-3 'direction contains the interfaces for the restriction endonucleases Smal, BamHI, Nhel and Ascl and the oligonucleotide primer SEQ ID NO: 2 in 5'-3' direction Interfaces for the restriction endonucleases Xbal, Xhol, Notl and Dral.
• the PCR reaction was carried out according to the standard method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, Academic Press (1990)) with PfuTurbo Polymerase (Stratagene, La Jolla, USA).
• the resulting DNA fragment with a size of approximately 2.1 kb was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the blunt ends of the DNA fragment were ligated together using the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation approach was carried out using standard methods as described in Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, described (1989)), transformed into competent E.coli XL-1Blue (Stratagene, La Jolla, USA).
• a selection for plasmid-carrying cells was carried out by the Plating on LB agar containing ampicillin (50 ⁇ g / ml) (Lennox, 1955, Virology, 1: 190) was achieved.
• the plasmid DNA of an individual clone was isolated using the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions and checked by restriction digestion.
• the plasmid obtained in this way is named pCLiKI.
• a kanamycin resistance cassette was amplified with the oligonucleotide primers SEQ ID NO: 3 and SEQ ID NO: 4.
• the oligonucleotide primer SEQ ID NO: 3 in the 5'-3 'direction contains the interfaces for the restriction endonucleases Xbal, Smal, BamHI, Nhel and the oligonucleotide primer SEQ ID NO: 4 in the 5'-3' direction Interfaces for the restriction endonucleases Ascl and Nhel.
• the PCR reaction was carried out according to the standard method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, Academic Press (1990)) with PfuTurbo Polymerase (Stratagene, La Jolla, USA).
• the linearized vector (approx. 2.1 kb) was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions. This vector fragment was ligated using the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer with the cut PCR fragment and the
• the plasmid DNA of an individual clone was isolated using the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions and checked by restriction digestion.
• the plasmid obtained in this way is named pCLiK2.
• the vector pCLiK2 was cut with the restriction endonuclease Dral (New England Biolabs, Beverly, USA). After electrophoresis in a 0.8% agarose gel, an approximately 2.3 kb vector fragment was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions. This vector fragment was religated using the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation approach was carried out according to standard methods as described in Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, described 1989)), transformed into competent E.coli XL-1Blue (Stratagene, La Jolla, USA). Selection for plasmid-bearing cells was achieved by plating on LB agar containing kanamycin (20 ⁇ g / ml) (Lennox, 1955, Virology, 1: 190).
• the plasmid DNA of an individual clone was isolated using the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions and checked by restriction digestion.
• the plasmid obtained in this way is named pCLiK3.
• the origin of replication pHM1519 was amplified with the oligonucleotide primers SEQ ID NO: 5 and SEQ ID NO: 6.
• SEQ ID NO: 6 5'-GAGAGGGCGGCCGCTCAAGTCGGTCAAGCCACGC-3 '
• the oligonucleotide primers SEQ ID NO: 5 and SEQ ID NO: 6 contain interfaces for the restriction endonuclease Notl.
• the PCR M / 43138 The reaction was carried out according to the standard method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, Academic Press (1990)) with PfuTurbo Polymerase (Stratagene, La Jolla, USA). The resulting DNA fragment with a size of approximately 2.7 kb was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, 5 Freiburg) according to the manufacturer's instructions.
• the DNA fragment was cut with the restriction endonuclease Notl (New England Biolabs, Beverly, USA) and then purified again with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the vector pCLiK3 was also cut with the restriction endonuclease Not1 and dephosphorylated with alkaline phosphatase (I (Röche Diagnostics, Mannheim)) according to the manufacturer. After electrophoresis in a 0.8% agarose gel, the linearized vector (approx. 2.3 kb) was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• This vector fragment was ligated with the aid of the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to 5 information from the manufacturer with the cut PCR fragment and the ligation approach according to standard methods as described in Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, described (1989)), transformed into competent E.coli XL-1Blue (Stratagene, La Jolla, USA).
• a selection for plasmid-bearing cells was carried out by plating on LB agar containing kanamycin (20 ⁇ g / ml) (Lennox, 1955, Virology,
• the plasmid DNA of an individual clone was isolated using the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions and checked by restriction digestion.
• the plasmid obtained in this way is given the name pCLiK5.
• the vector pCLiK5 was cut with the restriction endonucleases Xhol and BamHI (New England Biolabs, Beverly, USA) and dephosphorylated with alkaline phosphatase (I (Röche Diagnostics, Mannheim)) according to the manufacturer. After electrophoresis in a 0.8% agarose gel, the linearized vector (approx. 5.0 kb) was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• This vector fragment was ligated using the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer's instructions with the synthetic double-stranded DNA fragment and the ligation approach according to standard methods as described in Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, described (1989)), transformed into competent E.coli XL-1 Blue (Stratagene, La Jolla, USA). Selection for plasmid-bearing cells was achieved by plating on LB agar containing kanamycin (20 ⁇ g / ml) (Lennox, 1955, Virology, 1: 190).
• the plasmid DNA of an individual clone was isolated using the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions and checked by restriction digestion.
• the plasmid obtained in this way is named pCLiK5MCS.
• Sequencing reactions were carried out according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt).
• the resulting plasmid pCLiK5MCS is listed as SEQ ID NO: 9.
• SEQ ID NO: 10 5'-GAGAGCGGCCGCCGATCCTTTTTAACCCATCAC-3 '
• the oligonucleotide primers SEQ ID NO: 10 and SEQ ID NO: 11 contain interfaces for the restriction endonuclease Notl.
• the PCR reaction was carried out according to the standard method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, Academic Press (1990)) with PfuTurbo Polymerase (Stratagene, La Jolla, USA).
• the DNA fragment with a size of approximately 1.9 kb obtained was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the DNA fragment was cut with the restriction endonuclease Notl (New England Biolabs, Beverly, USA) and then purified again with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the vector pCLiK5MCS was also cut with the restriction endonuclease Not1 and dephosphorylated with alkaline phosphatase (I (Röche Diagnostics, Mannheim)) according to the manufacturer. After electrophoresis in a 0.8% agarose gel, an approximately 2.4 kb vector fragment was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• This vector fragment was ligated with the aid of the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer with the cut PCR fragment and the ligation approach according to standard methods as described in Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, described (1989)), transformed into competent E.coli XL-1Blue (Stratagene, La Jolla, USA). Selection for plasmid-bearing cells was achieved by plating on LB agar containing kanamycin (20 ⁇ g / ml) (Lennox, 1955, Virology, 1: 190).
• the plasmid DNA of an individual clone was isolated using the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions and checked by restriction digestion.
• the plasmid thus obtained is given the name pCLiK5MCS integrative sacB.
• the resulting plasmid pCLiK5MCS integrative sacB is listed as SEQ ID NO: 12.
• the fragment was cleaved with the restriction enzymes Mlu I and Sma I (Röche Diagnostics, Mannheim), which were introduced via the PCR oligonucleotide primers, and separated by gel electrophoresis.
• the DNA fragment was then purified from the agarose using GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg).
• the vector pCLiK5MCS, SEQ ID NO: 9 was also cleaved with the restriction enzymes Sma I and Mlu I and dephosphorylated with alkaline phosphatase I (Röche Diagnostics, Mannheim) according to the manufacturer.
• Vector and DNA fragment were ligated with T4 DNA ligase (Amersham Pharmacia, Freiburg) and according to standard methods as described in Sambrook et al. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, described, transformed into E.coli XL-1Blue (Stratagene). M / 43138
• the plasmid DNA was prepared using methods and materials from Quiagen. Sequencing reactions were carried out according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt).
• the resulting plasmid pCLiK5MCS / metKwt is listed as SEQ ID NO: 15.
• the directed mutagenesis of the metK gene from C. glutamicum was carried out using the QuickChange Kit (from Stratagene) and according to the manufacturer's instructions.
• the mutagenesis was carried out in the plasmid pCLiK5MCS / metKwt, SEQ ID NO: 15.
• the following oligonucleotide primers were synthesized for the replacement of cysteine 94 from SEQ ID NO: 16 for alanine 94:
• SEQ ID NO: 15 the use of these oligonucleotide primers leads to an exchange of the nucleotides in position 1056 (from C to G) and 1057 (from A to C).
• the resulting amino acid exchange Cys94Ala in the metK gene was confirmed by sequencing reactions after transformation and plasmid preparation.
• the plasmid was named pCLiK5MCS / metKC94A and is listed as SEQ ID NO: 19.
• C. glutamicum strains which were transformed either with the plasmid pCLiK5MCS / metKwt, SEQ ID NO: 15, or with the plasmid pCLiK5MCS / metKC94A, SEQ ID NO: 19, were in BHI / glucose medium (37 g / l Brain Heart Infusion finished medium, Difco, 10 mM (NH 4 ) 2 SO 4 , 4% glucose) at 30 ° C up to an OD 60 o of 20. The cells were centrifuged at 4 ° C and the pellet was washed with cold physiological saline washed.
• BHI / glucose medium 37 g / l Brain Heart Infusion finished medium, Difco, 10 mM (NH 4 ) 2 SO 4 , 4% glucose
• Reaction batches of 100 ⁇ l with 100 mM Tris pH 8.0, 100 mM KCI, 20 mM MgCl 2 , 1.2 mM L-methionine, 10 mM ATP, 1 ⁇ l 35 SL-methionine, corresponding to 15.15 ⁇ Ci (Amersham SJ204, spec. Activity 1 Ci / ⁇ mol) and H 2 O ad 100 ⁇ l were started with 100 ⁇ g of the respective protein lysate 5 and incubated at 37 ° C. At the times 0, 5, 10, 20, 30 and 60 minutes, 10 ⁇ l aliquots of the reaction mixture were removed and stopped on ice with 20 ⁇ l 50 mM EDTA.
• the rate of S-adenosylmethionine formation can be determined from the increase in built-in radioactivity per unit of time. Its unit is ⁇ mol 10 S-adenosylmethionine / min * mg protein. This rate can be compared between wild type enzyme and mutant enzyme.
• M / 43138 ID NO: 19 were transformed as follows.
• the S-adenosylmethionine content in the supernatant was determined by HPLC (lonospher 5C cation exchange column, 10 ⁇ l injection volume; eluent: 70% vol / vol 0.2 M ammonium formate pH 4.0 30% vol / vol methanol; UV detection 260 nm; 40 ° C; retention time 8.5 min.).
• the metK C94A sequence from SEQ ID NO: 19 was first cloned integratively into pCLiK5MCS sacB (SEQ ID NO: 12).
• the plasmid pCLiK5MCS / metKC94A (SEQ ID NO: 19) was cleaved with the restriction endonucleases Bgl II and Xho I (from NEB, Schwalbach).
• the resulting 1962 base pair fragment was purified as described in Example 3.
• the vector pCLiK5MCS integrative sacB was also cleaved with Bgl II and Xhol and purified as described in Example 3.
• Vector and fragment were ligated as described in Example 3, transformed into E.coli XL-1Blue.
• the plasmid was purified and confirmed after sequencing.
• the resulting plasmid pCLiK5MCS integrative sacB / metKC94A is listed as SEQ ID NO: 20.
• the plasmid pCLiK5MCS integrative sacB / metKC94A was in C. glutamicum KFCC10065 by means of electroporation as in Liebl, et al. (1989) FEMS Microbiology Letters 53: 299-303. Modifications to the protocol are described in DE 10046870. The chromosomal arrangement of the metK locus of individual transformants was determined using standard methods by Southemblot and hybridization as described in Sambrook et al. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor.
• the sacB / metKC94A integratively contained sacB / metKC94A in the vector pCLiK5MCS converts sucrose into a toxic product
• only those colonies can grow which have deleted the sacB gene by a second homologous recombination step between the wild-type metK gene and the mutated metKC94A gene.
• the homologous recombination either the wild-type gene or the mutated gene together with the sacB gene can be deleted. If the sacB gene is removed together with the wild-type gene, a mutant transformant results.
• the strain KFCC10065metKC94A prepared in Example 6 was grown on an agar plate with BHI medium (Difco) for 2 days at 30 ° C.
• the grown cells were suspended in saline from the agar plate and transferred to medium II with an OD 600 nm of 1.5.
• Medium II was composed as follows.
• the medium thus prepared was adjusted to pH 7.8 with NH 4 OH and sterilized at 120 ° C. for 30 minutes.
• Medium MB were prepared separately, sterilized by filtration and added to Medium IIA. Both components Ha and MB together result in Medium II.
• Methionine formed in the culture broth was determined using Agilent's amino acid determination method on an Agilent 1100 Series LC System HPLC.
• a pre-column derivatization with ortho-phthalaldehyde allows the quantification of the amino acids formed, the separation of the amino acids takes place on a Hypersil AA column (Agilent).

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